Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are techniques for amplifying any desired nucleic acid sequence (target) contained in a nucleic acid or mixture thereof. In PCR, a pair of primers are employed in excess to hybridize at the outside ends of complementary strands of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules. PCR is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202.
LCR is an alternate mechanism for target amplification. In LCR, two sense (first and second) probes and two antisense (third and fourth) probes are employed in excess over the target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being positioned so that the primary probes can be ligated into a fused product. Further, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar ligatable fashion. If the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first instance. Once the fused strand of sense and antisense probes are separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product. The fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described in EP-A-320,308, hereby incorporated by reference. Other aspects of LCR technique are disclosed in EP-A-439,182, to Backman, K. C. et al., hereby incorporated by reference.
Ironically, the biggest virtue of PCR and LCR techniques also poses a significant problem for diagnostic assays. Since both techniques exponentially amplify their targets, they are so sensitive to tiny amounts of target DNA that even ten molecules of oligonucleotide that arise from exogenous sources rather than from the sample itself can lead to a false positive result and mistyping. In the case of PCR, it has been found that if one round of PCR with 25-40 cycles is performed in a volume of 25 .mu.L, approximately 10,000 contaminating molecules are sufficient to produce a spurious band that can be detected with ethidium bromide. Sarkar, G. et al., BioTechniques, 10(5): 591-593 (1991). If nested PCR or booster PCR are used for applications in which DNA from 1-10,000 cells is the input, extreme caution is required because contamination with only one molecule may produce a spurious result. Id.
A primary source of exogenous templates leading to false positive amplifications are carryover products from previous PCR or LCR amplifications of the same target ("PCR products" or "LCR products" respectively) which become dispersed in the laboratory area and serve as templates in subsequent PCR or LCR amplification. PCR or LCR products may also contaminate physically proximate negative samples ("crossover contamination"). Besides contamination by PCR or LCR products, the reagents, samples, equipment, and the general laboratory area may also be contaminated by exogenous DNAs such as bacterial or viral DNAs. For example, PCR may register high levels of background amplification for samples containing water but no DNA, due to the presence of small fragments of DNA which may be produced by autoclaving viral culture material. Porter-Jordan, K. et al., J. Med. Virol., 30(2): 85-91 (1990). RNA contamination is a lesser problem because RNAases are commonly present in the environment. To avoid contamination in the case of PCR, it has been suggested that before adding template DNA and Taq DNA polymerase, individual reaction mixtures should be treated with DNasel or restriction endonucleases that cut internal to the pair of amplification primers. Furrer, B. et al., Nature (London), 346: 6282-324 (1990). This treatment will inactivate contaminating nucleic acid sequences, e.g., render them biologically inactive to polymerase or ligase.
Certain other simple steps are used in PCR to avoid all the above contaminations. They include: (1) physically isolating PCR preparations and products; (2) autoclaving solutions where possible; (3) dividing reagents into aliquots to minimize the number of repeated samplings necessary; (4) using of disposable gloves; (5) avoiding splashes; (6) using disposable replacement pipettes; (7) "premixing" reagents where possible; (8) adding DNA last; and (9) choosing positive and negative controls carefully. Kwok S., et al, Nature, 339, 6221, 237-38 (1989).
Additionally, several more specific methods have been described to eliminate carryover DNA target products. These methods include ultraviolet (UV) irradiation (e.g., Amino, et al. Nature, 345: 773 (1990), gamma irradiation (Deragan, et al., Nucl. Acids Res., 18: 6149 (1990)), psoralen treatment (Jinno, et al., Nucl. Acids Res., 18: 6739 (1990)), and the use of uracil-N-glycosylase treatment of dU-containing DNA (Longo, et al., Gene, 93: 125 (1990); Package Insert, GeneAmp PCR Carry-Over Prevention Kit, Perkin-Elmer, 1990). UV or gamma irradiation or psoralen treatment in conjunction with UV irradiation is of limited efficiency. The efficiency of such sterilization is dependent on the size and specific sequence of the product. In the case of UV irradiation, for example, longer PCR products (more than 700 base pairs) appear to be more susceptible to UV irradiation than do shorter PCR products (less than 250 base pairs) (Amino, et al., supra.). Additionally, insufficient data have been generated regarding irradiation effect on the chemical integrity of the primers.
In PCR, the carryover contamination problem is solved to some extent, by replacing normal ribonucleoside triphosphates (rNTPs) or deoxyribonucleotide triphosphates (dNTPs) with an exogenous nucleotide that is not present in natural nucleic acid. (See European Patent Application publication no. 0401037, published Dec. 5, 1990, to Hartley). The exo-sample nucleotides used are uracil N-glycosylase (UNG) and uridine triphosphate. The method works thus: the UNG cleaves the glycosidic bond between the base uracil and the sugar deoxyribose, but only when the 2'-deoxyuridine moiety is incorporated into the deoxyribonucleic acid (DNA). The enzyme does not act upon dUTP, free deoxyuridine, or RNA. Thus, if DNA containing 2'-deoxyuridine is introduced into a sample, the adventitious DNA will be cleaved if UNG is present in the medium, while the UNG will not affect the natural DNA. The cleaved DNA will not be a substrate for PCR. In the first cycle of PCR, the UNG will be denatured, and so will not act on the UTP which is incorporated into the growing oligonucleotide chain as part of PCR. Importantly, this method does not address the problem of carryover contamination from native DNA which has the base thymidine instead of uridine, as thymidine is not a substrate for UNG.
Apart from the above PCR investigation, researchers in the field of gene activity and inactivation in live cells also work with the cleavage of DNA. However, since a gene function can be destroyed or altered through even a single cleavage in the nucleotide sequence, the emphasis in this field, site directed mutagenesis, is just to cleave or nick a DNA sequence, preferably at a specific site, in order to observe the resulting effect on the gene function or cell behavior. Thus, the intent is not to totally destroy or inactivate all the DNA sequences, unlike that of PCR or LCR.
Metal chelate complexes have been used in conjunction with oxidizing and reducing agents to damage DNA. The art is replete with findings that reducing agents are required for DNA damage, if metal chelates are used to cleave the DNA. For example, Que et al indicate that the degradation of Escherichia coli DNA by 1,10-phenanthroline requires Cu(lI), oxygen, and a reducing agent. Que, B. G. et al., Biochem., 19(26): 5987-5991 (1980).
Similarly, Sigman (Acc. Chem. Res., 19: 180-186, 1986) and Goldstein et al. (J. Am. Chem. Soc., 108: 2244-2250 (1986); J. Free Radicals in Biology & Medicine, 2: 3-11, 1986) disclose the use of a reducing agent, copper bis(1,10-phenanthroline), and hydrogen peroxide to cleave DNA.
Apart from interfering with LCR and PCR results, undesirable nucleotide sequences also pose a problem in bioprocesses and the resulting bioproducts, for example, in cell-culture-derived recombinant proteins. These contaminating nucleotides can be of viral, fungal, or bacterial origins, such as endogenous and adventitious retroviruses. Knight, P., Bio/Tech., 6(12): 1373-73 (1988). These contaminating nucleotides are health hazards especially if the bioproducts are used in vivo. For example, viral DNA can potentially infect the recipient of a transfused recombinant protein.
The current modes of DNA removal consist of DNAse treatment, polymin P precipitation and anion exchange liquid chromatography separation. The major problem with DNAse treatment is that inhibitors present in the medium can block or slow action of the enzyme. Further, if active DNAase is still present in the recombinant protein preparation that is introduced into a patient, it would seriously jeopardize the patient's health as the DNAase would permeate the patient's cells and destroy the DNA therein. The shortcoming of polymin P precipitation is that it can remove some, but not all, of the endogenous DNA. Finally, anion exchange liquid chromatography carries with it the possibilities of incomplete separation of highly charged protein from DNA, and carryover of sample.